The regulation of mitochondrial transport in neurons is of particular interest due to the unique morphology of neurons and their high energy demands. Defective mitochondrial trafficking can induce changes in mitochondrial fusion/fission dynamics, local bioenergetic profiles, reactive oxygen species (ROS) production, and Ca2+ homeostasis. Several studies have associated altered mitochondrial transport with axon degeneration and the pathogenesis of neurodegenerative disorders, thus demonstrating the importance of the regulation of mitochondrial transport. Previously, the mitochondrial transport adaptor protein, Milton, was shown to interact with O-GlcNAc transferase, the enzyme that catalyzes the addition of O- GlcNAc to proteins. O-GlcNAc is a dynamic post-translational modification that modulates a wide variety of cellular processes and is thought to be a metabolic sensor. Recent work in the Schwarz lab demonstrated that acute changes in glucose levels can regulate mitochondrial trafficking in neurons through the O-GlcNAcylation of Milton1. This leads to the question of whether altered O-GlcNAc dynamics in neurons under chronic hyperglycemic conditions is involved in the pathogenesis of diabetic neuropathy, and in particular whether dysregulation of mitochondrial transport is a contributing factor. The goals of this proposal are: 1. To investigate changes in the O-GlcNAc levels of Milton1 in hyperglycemic neurons. 2. To associate changes in mitochondrial and axonal health with changes in mitochondrial motility 3. To determine the physiological relevance of Milton1 O-GlcNAcylation in vivo. 4. To determine the role of O- GlcNAcylation of Milton1 in mouse models of diabetic neuropathy. By addressing these questions, this proposal aims to further elucidate the significance of dynamic O-GlcNAc levels and the regulation of mitochondrial motility in mammalian neurons under conditions of hyperglycemia and diabetic neuropathy. Overall, the completion of the proposal will provide broad insight on the role of mitochondria in axon degeneration and neuropathy as well as further characterization of a novel regulatory role of the O-GlcNAc modification.